ROBUST PROTEIN SEALANTS FROM MUSSEL BYSSUS

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The byssus, an external tendon-like structure in marine mussels, has at least two scientifically intriguing properties: A. It mediates the strong and opportunistic adhesion that these animals exhibit on a wide surfaces in the sea (metal, glass, mineral, polymethylmethacrylate, etc), and B. It possesses a physical and chemical robustness that exceeds the robustness of its individual constituents such as collagen and mussel adhesive protein (MAP). This robustness is apparently imparted by the quinone tanning of a colloidal sealant with which every portion of the byssus is coated (thickness 10 micromoles) prior its release into the sea water. Because of this sealant's resistance to microbial attack, its affinity for collagenous tissues, and its broad stability to acids, bases, organics, and heat, a better understanding of its formation, composition and stabilization could contribute significantly to the inspired design of medical/dental sealants. This is particularly appropriate for the protection of collagen-containing tissues such as dentin, bone, articular cartilage, and tendon that undergo a variety of degenerative processes. The experimental goal of this proposal is to isolate granules containing sealant precursors from the accessory gland of the mussel (Mytilus edulis) foot. This will be done either by inducing degranulation or tissue homogenization followed by differential centrifugation. Isolated granules will be lysed and contents will be analyzed by assays for enzymes (catecholoxidase, phosphatase), 3,4- dihydroxyphenylalanine (DOPA), by polyacrylamide gel electrophoresis and western blotting using available polyclonal antibodies (anti-catecholoxidase and anti-MAP) as probes. The composition, N- terminal sequence, and relative concentration of each of the granule proteins will be determined by traditional protein chemistry. Mono- or polyclonal antibodies will be prepared to each of the novel proteins, and location of each of these proteins in the accessory gland granule as well as in the actual byssal sealant will be attempted by immunofluorescent or colloidal gold techniques. The ability of any of the proteins to protect collagen will be assessed using an in vitro assay containing the putative shielding agent, a soluble collagen (type II, calf skin) and clostridial collagenase at pH 8.0 (0.1 M borate). Finally, the mechanism of stabilization of the colloidal sealant by quinone-tanning will be probed by adding a strong nucleophile, C-14 glycylmethylester, to activated presealant granules. In this way, the resident catecholoxidase can form peptidyl o-quinones in MAP or some similar protein without leading to cross-links. Modified, but still soluble, MAP can then be recovered and characterized.